The goal of the project is to develop a therapeutic capable of sterilizing recalcitrant chronic infections such as deep-seated abscess, osteomyelitis, endocarditis, and biofilms of indwelling devices. Many of these infections are essentially untreatable and lead to substantial morbidity and mortality. In many cases, recalcitrance of an infection is not caused by drug resistance. Rather, a slow-growing biofilm population harbors stationary phase and dormant persister cells that are highly tolerant to killing by antimicrobials. When antibiotic concentration drops, these cells can grow and repopulate the biofilm. In Gram-positive S. aureus, it appears that a stationary culture is made mostly of drug tolerant cells which are virtually insensitive to killing by traditional antibiotics. There are many independent, redundant mechanisms of persister formation, and these specialized survivor cells do not have a realistic target which could be exploited for drug development. In order to act, all existing bactericidal antibiotics require active targets which they corrupt. We reasoned that persisters could be killed if a small molecule could simultaneously activate and corrupt a cellular target. We find that acyldepsipeptide (ADEP) activates the ClpP protease in dormant persisters, forcing the cell to self-digest. In order to diminish resistance development, ADEP was combined with rifampicin. The combination completely sterilized a deep- seated biofilm infection of S. aureus in a neutropenic mouse model after a single dose. The best conventional antibiotics, alone or in combination, had very little effect. This model emulates the most difficult to treat chronic infection in immunocompromised patients. In this project, we will identify the most promising sterilizing combinations of drugs. Combinations will be evaluated for PK, PD, and efficacy using a deep-seated neutropenic thigh model of MRSA infection, and in an in vivo biofilm tissue-cage model. Once validated, the combination therapeutic will enter into preclinical investigation in Phase II, leading to an IND, and subsequent clinical trials of the drug.